The gut requires the presence of intrinsic neurons for motility and absorption/secretion to occur. The intrinsic or enteric neurons develop from neural crest cells that migrate from the neural tube and enter the gut. Within the gut these crest cells migrate, aggregate, proliferate, and differentiate into neurons which interact to form the neural circuitry of the gut. Within the gut these crest cells migrate, aggregate, proliferate, and differentiate into neurons which interact to form the neural circuitry of the gut. The long-term objective of this study is to elucidate the mechanisms which regulate the formation of the enteric nervous system. The formation and maintenance of the enteric nervous system are essential to the formal function of the gut. A knockout of the gene for Glial Derived Neurotrophic Factor (GDNF) and for its receptor Ret results in similar phenotypes. These mice show a small number of neurons in the foregut but none in the small and large intestine. The overall objective of this proposal is to discover the critical role GDNF plays in the development of enteric nervous system. Our first aim is to determine when and where GDNF is made in the gut. We will use in situ hybridization, immunocytochemistry, ELISA, and microsurgery to localize and quantitate GDNF expression. Our second goal is to determine when and where independent cells are able to colonize the intestine. Immunocytochemistry and in situ hybridization will be used to examine the appearance of Ret. To determine whether Ret independent cells (which do not require GDNF for survival) are able to colonize the gut, gut from GDNF null mice will be explanted with qual and mouse truncal neural crest cells. This experiment will reveal whether Ret dependence is determined before or after neural crest are critically dependent on GDNF. The enteric nervous system is formed in 3 stages: neural crest migrate, neural crest enter the gut and differentiate, neural crest as neurons and glia. We will evaluate the effects of GDNF on these stages. We will culture cells from each of these stages and immunostain them to assess changes in proliferation, differentiation, and survival in the presence of GDNF. We will also determine whether GDNF is necessary for the maintenance of the adult enteric nervous system by culturing adult neurons. Completion of this work will provide insight into the role of GDNF in the formation and possibly the maintenance of the enteric nervous system. This information will advance our knowledge of how the defects in GDNF or Ret expression in the development or in the adult might affect the structure and function of the enteric nervous system.